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Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles

Nature Neuroscience volume 13, pages 845–851 (2010)Cite this article

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Abstract

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.

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Figure 1: Dynamin I is an in vitro substrate for GSK3β only after priming by cdk5.
Figure 2: Dynamin I is selectively phosphorylated on Ser-774 by GSK3 in neurons.
Figure 3: GSK3-dependent protein rephosphorylation is required for synaptic vesicle retrieval during high-intensity stimulation.
Figure 4: Activity-dependent requirement for GSK3-dependent protein rephosphorylation in synaptic vesicle retrieval.
Figure 5: GSK3-dependent protein rephosphorylation is required for bulk endocytosis of large dextrans.
Figure 6: GSK3-dependent protein rephosphorylation is required for HRP uptake into endosomes, but not synaptic vesicles.
Figure 7: GSK3β phosphorylation of Ser-774 on dynamin I is required for bulk endocytosis of large dextrans.
Figure 8: Inhibition of GSK3 relieves HFS depression of EPSCs in intact slices.

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Acknowledgements

We wish to thank A. Prescott and J. James (both University of Dundee) for excellent technical assistance. This work was supported by grants from the Wellcome Trust (Ref: 070569 & 084277), Epilepsy Research UK (0503), Cancer Institute NSW, The Ramaciotti Foundation, and the Australian National Health and Medical Research Council. C.S. is supported by the Alzheimer's Research Trust (ART/PG2007/4) and the MRC (88158). N.B. is supported by the Danish Agency for Science, Technology and Innovation (grant 274-08-0066). A.R.C. is supported by an Australian NH&MRC Peter Doherty Fellowship (454886).

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  1. Adam R Cole

    Present address: Present address: Neurosignaling Group, Garvan Institute for Medical Research, Sydney, Australia.,

Authors and Affiliations

  1. Centre for Integrative Physiology, George Square, University of Edinburgh, Edinburgh, Scotland, UK

    Emma L Clayton, Karen J Smillie, Timothy O'Leary, Giselle Cheung, David J Wyllie & Michael A Cousin

  2. Children's Medical Research Institute, The University of Sydney, Wentworthville, Sydney, Australia

    Nancy Sue, Nicolai Bache & Phillip J Robinson

  3. Biomedical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland, UK

    Adam R Cole & Calum Sutherland

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Contributions

M.A.C. and P.J.R. designed the experimental protocols and led the project. C.S. provided the concept for GSK3 phosphorylation of dynamin I and the selective GSK3 antagonists. M.A.C., A.R.C., C.S., D.J.W. and P.J.R. wrote the manuscript. E.L.C., M.A.C., G.C. and K.J.S. performed all experiments using intact neurons and subsequent analysis. D.J.W. and T.O. designed and performed experiments with hippocampal slices. N.S. and N.B. performed all in vitro phosphorylation experiments and subsequent analysis. A.R.C. provided preliminary experiments for the project.

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Correspondence to Michael A Cousin.

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Clayton, E., Sue, N., Smillie, K. et al. Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles. Nat Neurosci 13, 845–851 (2010). https://doi.org/10.1038/nn.2571

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